Evaluation of ELP, collagen and laminin hydrogel composites to support in vitro differentiation of hADSCs toward adipocytes and β-cells

Drug discovery typically involves studies on animal models as proxies for human beings followed by three phases of clinical studies, which result in frequent late rejections due to interspecies differences, present moral issues and requires extensive administration. Hence, large efforts are made to develop in vitro drug screening assays based on human cells, which provide detailed information on intracellular processes and enable high-throughput analysis of multiple drugs in parallel. Assays mimicking metabolic diseases such as diabetes and obesity are of particular interest, due to their increasing prevalence and due to important interspecies differences in metabolic pathways. However, it becomes increasingly clear that cells grown in vitro on conventional flat surfaces have difficulties maintaining their native, mature functionalities and eventually develop artificial phenotypes. For that reason, we have evaluated tissue-mimicking, protein-composite hydrogels for three-dimensional growth and differentiation of human adipocytes-derived stem cells (hADSCs) toward (i) adipocytes and (ii) β-cells. The proteins collagen I, laminin isoforms 411 and 511 and recombinant-engineered elastin-like proteins (ELP) have been investigated in detail, individually as well as in mixes. Their different amino-acid sequences coding for cell attachment/interaction, attractive material properties (i.e. soft and hydrated) together with the three-dimensional encapsulation are intended to mimic the native microenviroment, and thereby drive the differentiation and cellular maturation. The metabolic activity, viability and function of the differentiated cells were analyzed with cellular assays. Further, the tissue-mimicking samples were evaluated with non-linear microscopy; multi-photon excitation fluorescence (MPEF, autofluorescence and stains), coherent anti-Stokes Raman scattering (CARS, cellular lipid content) and second harmonic generation (SHG, collagen fibers). The assays and MPEF microscopy revealed that cells do not thrive in the ELP hydrogels crosslinked with amine-reactive THPC, probably due to cytotoxicity of the crosslinker and as the hydrogel seems not to permit cellular expansion. In contrast, cells in collagen and collagen/ELP matrices were successfully differentiated into adipocyte-like cells, confirmed by the presence of excessive amounts of lipid stores co-localized with perilipin. Insulin-positive cells were found in the collagen and collagen/laminin matrices. Hence, different collagen mixes seem to be promising hydrogel materials for three-dimensional growth of hADSCs for metabolic drug screening.

BibTeX @mastersthesis{Åström2015,author={Åström, Maria and Dahlén, Robin},title={Evaluation of ELP, collagen and laminin hydrogel composites to support in vitro differentiation of hADSCs toward adipocytes and β-cells},abstract={Drug discovery typically involves studies on animal models as proxies for human beings followed by three phases of clinical studies, which result in frequent late rejections due to interspecies differences, present moral issues and requires extensive administration. Hence, large efforts are made to develop in vitro drug screening assays based on human cells, which provide detailed information on intracellular processes and enable high-throughput analysis of multiple drugs in parallel. Assays mimicking metabolic diseases such as diabetes and obesity are of particular interest, due to their increasing prevalence and due to important interspecies differences in metabolic pathways. However, it becomes increasingly clear that cells grown in vitro on conventional flat surfaces have difficulties maintaining their native, mature functionalities and eventually develop artificial phenotypes. For that reason, we have evaluated tissue-mimicking, protein-composite hydrogels for three-dimensional growth and differentiation of human adipocytes-derived stem cells (hADSCs) toward (i) adipocytes and (ii) β-cells. The proteins collagen I, laminin isoforms 411 and 511 and recombinant-engineered elastin-like proteins (ELP) have been investigated in detail, individually as well as in mixes. Their different amino-acid sequences coding for cell attachment/interaction, attractive material properties (i.e. soft and hydrated) together with the three-dimensional encapsulation are intended to mimic the native microenviroment, and thereby drive the differentiation and cellular maturation. The metabolic activity, viability and function of the differentiated cells were analyzed with cellular assays. Further, the tissue-mimicking samples were evaluated with non-linear microscopy; multi-photon excitation fluorescence (MPEF, autofluorescence and stains), coherent anti-Stokes Raman scattering (CARS, cellular lipid content) and second harmonic generation (SHG, collagen fibers). The assays and MPEF microscopy revealed that cells do not thrive in the ELP hydrogels crosslinked with amine-reactive THPC, probably due to cytotoxicity of the crosslinker and as the hydrogel seems not to permit cellular expansion. In contrast, cells in collagen and collagen/ELP matrices were successfully differentiated into adipocyte-like cells, confirmed by the presence of excessive amounts of lipid stores co-localized with perilipin. Insulin-positive cells were found in the collagen and collagen/laminin matrices. Hence, different collagen mixes seem to be promising hydrogel materials for three-dimensional growth of hADSCs for metabolic drug screening.},publisher={Institutionen för biologi och bioteknik, Kemisk biologi, Molekylär mikroskopi, Chalmers tekniska högskola},place={Göteborg},year={2015},keywords={3D scaffold, Adipocytes, β-cells, collagen, diabetes, ELP, hADSCs, laminin, non-linear microscopy, obesity},note={99},}

RefWorks RT GenericSR ElectronicID 217875A1 Åström, MariaA1 Dahlén, RobinT1 Evaluation of ELP, collagen and laminin hydrogel composites to support in vitro differentiation of hADSCs toward adipocytes and β-cellsT2 Evaluation of ELP, collagen and laminin hydrogel composites to support in vitro differentiation of hADSCs toward adipocytes and β-cellsYR 2015AB Drug discovery typically involves studies on animal models as proxies for human beings followed by three phases of clinical studies, which result in frequent late rejections due to interspecies differences, present moral issues and requires extensive administration. Hence, large efforts are made to develop in vitro drug screening assays based on human cells, which provide detailed information on intracellular processes and enable high-throughput analysis of multiple drugs in parallel. Assays mimicking metabolic diseases such as diabetes and obesity are of particular interest, due to their increasing prevalence and due to important interspecies differences in metabolic pathways. However, it becomes increasingly clear that cells grown in vitro on conventional flat surfaces have difficulties maintaining their native, mature functionalities and eventually develop artificial phenotypes. For that reason, we have evaluated tissue-mimicking, protein-composite hydrogels for three-dimensional growth and differentiation of human adipocytes-derived stem cells (hADSCs) toward (i) adipocytes and (ii) β-cells. The proteins collagen I, laminin isoforms 411 and 511 and recombinant-engineered elastin-like proteins (ELP) have been investigated in detail, individually as well as in mixes. Their different amino-acid sequences coding for cell attachment/interaction, attractive material properties (i.e. soft and hydrated) together with the three-dimensional encapsulation are intended to mimic the native microenviroment, and thereby drive the differentiation and cellular maturation. The metabolic activity, viability and function of the differentiated cells were analyzed with cellular assays. Further, the tissue-mimicking samples were evaluated with non-linear microscopy; multi-photon excitation fluorescence (MPEF, autofluorescence and stains), coherent anti-Stokes Raman scattering (CARS, cellular lipid content) and second harmonic generation (SHG, collagen fibers). The assays and MPEF microscopy revealed that cells do not thrive in the ELP hydrogels crosslinked with amine-reactive THPC, probably due to cytotoxicity of the crosslinker and as the hydrogel seems not to permit cellular expansion. In contrast, cells in collagen and collagen/ELP matrices were successfully differentiated into adipocyte-like cells, confirmed by the presence of excessive amounts of lipid stores co-localized with perilipin. Insulin-positive cells were found in the collagen and collagen/laminin matrices. Hence, different collagen mixes seem to be promising hydrogel materials for three-dimensional growth of hADSCs for metabolic drug screening.PB Institutionen för biologi och bioteknik, Kemisk biologi, Molekylär mikroskopi, Chalmers tekniska högskola,PB Institutionen för biologi och bioteknik, Kemisk biologi, Molekylär mikroskopi, Chalmers tekniska högskola,LA engLK http://publications.lib.chalmers.se/records/fulltext/217875/217875.pdfOL 30